Phenotypic reversion of pancreatic carcinoma cells

ABSTRACT

The present invention provides peptides (including analogs and derivatives thereof) corresponding to residues 96-110 and 35-47 of ras-p21, which peptides have attached thereto a membrane-penetrating leader sequence. The subject peptides, analogs and derivatives thereof are useful in treatment of cancers and have been shown to induce phenotypic reversion of pancreatic cancer cells to non-cancerous cells. Pharmaceutical compositions comprising one or more subject peptides are also provided by the present invention. The present invention further provides replication incompetent Adenovirus (AdV) vectors comprising a promoter sequence and a nucleotide sequence encoding a subject peptide. Methods of treating cancer by administering one or more subject peptides, pharmaceutical compositions, and/or AdV vectors are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 13/677,876, filed Nov. 15,2012, which is a divisional of U.S. Ser. No. 12/488,209, filed Jun. 19,2009; which is a divisional of U.S. Ser. No. 11/825,242, filed Jul. 5,2007; which is a continuation application of U.S. Ser. No. 11/142,051,filed May 31, 2005, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/575,131, filed May 28, 2004, and U.S.Provisional Application Ser. No. 60/575,846, filed Jun. 1, 2004, both ofwhich are incorporated by reference herein.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The present invention was funded in part by NIH Grant ROI CA 42500; thegovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Oncogenic ras-p21 protein, but not its wild-type counterpart protein,induces malignant transformation of mammalian cell lines such as NIH 3T3cells (1) and has been implicated as a major causative factor in a highproportion of human solid tissue tumors (2). In Xenopus laevis oocytes,microinjection of oncogenic (containing Val in place of Gly 12), but notwild-type, p21 induces oocyte maturation (3). Insulin induces oocytematuration and requires activation of normal cellular ras-p²¹ (4).

Several agents that strongly block Val 12-p21-induced oocyte maturationhave virtually no effect on insulin-induced maturation (5). Among theseagents are specific peptides, identified from molecular modelingstudies, that correspond to effector domains from both ras-p21 itself,such as the 35-47, 96-110 and 115-126 sequences (5) and from some of itstarget proteins such as the ras-binding domain of raf (residues 97-110)(6-8) and the SOS guanine nucleotide exchange protein (residues994-1004) (9,10). These peptide domains were identified as those thatchange conformation in response to the presence of single oncogenicamino acid substitutions at positions 12 or 61 or multiple substitutionsat positions 10, 12 and 59 when the computed average structures forthese proteins either alone or in complex with target proteins weresuperimposed on that for the wild-type protein.

The finding that these peptides (in addition to other agents) blockoncogenic ras-p21 selectively indicates that the oncogenic proteininduces mitogenesis by pathways that may overlap with, but are alsodistinct from, pathways utilized by the wild-type protein. In studiesdesigned to identify pathway differences, it was found that, in oocytes,oncogenic but not insulin-activated wild-type ras-p21 interacts with thetranscriptional activating protein, jun and its kinase, jun kinase (JNK)(11,12), and requires the presence of protein kinase C (PKC) (13). Inthese studies, it was determined that the peptide whose sequencecorresponds to p21 residues 96-110, called PNC-2, blocks the interactionof Val 12-p21 with JNK (11,12) in a dose-response curve thatsuperimposes on that for its inhibition of Val 12-p21-induced oocytematuration (5).

Additionally, the peptide whose sequence corresponds to p21 residues35-47, called PNC-7, encompasses a domain of the protein implicated inits interacting with multiple targets including raf p74 protein, GTPaseactivating protein (GAP) and the guanine nucleotide exchange protein,SOS (reviewed in ref. 5). This peptide strongly inhibits c-raf-inducedoocyte maturation but has no effect on oocyte maturation induced by anoncogenic mutant raf lacking the ras binding domain (RBD) in its aminoterminal regulatory domain (14). Both PNC-2 and 7 appear to act ondifferent steps on the oncogenic ras-p21 signal transduction pathway.For example, PNC-2 but not PNC-7 interferes with Val 12-p21-JNKinteraction (11,12) while PNC-7 but not PNC-2 blocks signal transductionthrough c-raf (15).

Since various cancers involve expression of Val 12-p21 protein, as wellas other oncogenic proteins, it would be useful to be able to inhibitexpression of such proteins. For example, pancreatic cancer is a nearlyalways fatal disease with a median survival time of only 80-90 days fora patient diagnosed with the disease. Pancreatic cancer is one of themore lethal forms of cancer in numbers of patients killed in the U.S.Less than 4% of patients are alive 5 years from the time of diagnosis.The present invention provides peptides and pharmaceutical compositionscomprising such peptides which when administered to pancreatic cancercells, not only inhibit oncogenic Val 12-p21 but actually causecancerous cells to phenotypically revert to non-cancerous cells. Thepresent invention is therefore useful in treating various types ofcancers which express Val 12-p21 protein and/or other oncogenicproteins. Treatment of ras-induced tumors converts malignant masses intobenign ones, allowing for the halting of metastatic disease.

SUMMARY OF THE INVENTION

The present invention provides peptides comprising at least about tencontiguous amino acids of the amino acid sequence: YREQIKRVKDSDDVP (SEQID NO: 1), or an analog or derivative thereof, wherein said peptide,analog, or derivative thereof comprises a membrane-penetrating leadersequence attached thereto. Preferably, a peptide has the sequence setforth in SEQ ID NO:1.

The present invention also provides peptides comprising at least aboutten contiguous amino acids of the amino acid sequence: TIEDSYRKQVVID(SEQ ID NO:2) or an analog or derivative thereof wherein said peptide,analog, or derivative thereof comprises a membrane-penetrating leadersequence attached thereto. Preferably, a peptide has the sequence as setforth in SEQ ID NO:2.

The peptides of the present invention, including analogs and derivativesthereof, are useful in treating cancer. Preferably, a peptide, analog orderivative thereof as provided herein has the membrane-penetratingleader sequence located at the carboxy terminal end. In anotherpreferred embodiment, the leader sequence comprises predominantlypositively charged amino acid residues. Examples of leader sequences forpracticing the present invention include but are not limited topenetratin, Arg₈, TAT of HIV1, D-TAT, R-TAT, SV40-NLS,nucleoplasmin-NLS, HIV REV, FHV coat, BMV GAG, HTLV-II (REX), CCMV GAG,P22N, Lambda N, Delta N, yeast PRP6, human U2AF, human C-FOS, humanC-JUN, yeast GCN4, or p-vec.

Also provided by the present invention are pharmaceutical compositionscomprising at least one of the subject peptides or analogs orderivatives thereof comprising a membrane-penetrating leader sequenceadmixed with a pharmaceutically acceptable carrier.

The present invention also provides methods of treating a patientsuffering from cancer. The method comprises administering to saidpatient a therapeutically effective amount of at least one subjectpeptide, analog or derivative thereof comprising a membrane penetratingleader sequence. In another embodiment of the invention, there isprovided a method of treating a patient suffering from cancer byadministering to said patient a therapeutically effective amount of asubject pharmaceutical composition. Preferably, the cancer to be treatedis a ras-induced cancer.

In still another embodiment of the invention, there is provided areplication incompetent Adenovirus (AdV) vector comprising a promotersequence operably linked to a nucleotide sequence encoding a peptide,wherein the peptide comprises at least about ten contiguous amino acidsof the amino acid sequence: YREQIKRVKDSDDVP (SEQ ID NO: 1), or an analogor derivative thereof. A replication incompetent Adenovirus (AdV) vectorcomprising a promoter sequence operably linked to a nucleotide sequenceencoding a peptide, wherein the peptide comprises at least about tencontiguous amino acids of the amino acid sequence: TIEDSYRKQVVID (SEQ IDNO:2), or an analog or derivative thereof is also provided. Preferably,the nucleotide sequence further encodes a leader sequence attached tothe sequence set forth in SEQ ID NO: 1.2, or an analog or derivativethereof.

The present invention also provides a method of treating a patientsuffering from cancer by administering to the patient, a therapeuticallyeffective amount of a subject AdV vector. A method of inducingphenotypic reversion of cancerous cells to non-cancerous cells in asubject, by administering to the subject, a therapeutically effectiveamount of a subject AdV vector is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph of untreated ras-transformed pancreaticcancer (TUC-3) cells.

FIG. 1B is a photomicrograph of TUC-3 cells treated with X13-leaderpeptide for two weeks.

FIG. 1C is a photomicrograph of untreated pancreatic acinar (BMRPA1)cells at confluence.

FIG. 1D is a photomicrograph of BMRPA1 cells treated with PNC-2-leaderpeptide, showing no change in morphology or cell viability.

FIG. 2A. is a photomicrograph showing the effects of 100 μg/ml ofPNC-2-leader on TUC-3 cells after two weeks of treatment.

FIG. 2B is a photomicrograph showing the effects of 100 μg/ml ofPNC-2-leader on TUC-3 cells after one day of treatment. In the center ofthe figure, a focus of morphologically revertant cells is shown.

FIG. 2C is a photomicrograph showing the effects of 100 μg/mlPNC-7-leader peptide on TUC-3 cells after two weeks of treatment.

FIG. 3A is a photomicrograph taken one week after plating transfected(with PNC-2-expressing plasmid) viable TUC-3 cells in selective media.Foci of reversion can be observed (left and middle of figure). Remainingtransformed cells can be seen on the right side of the figure.

FIG. 3B is a photomicrograph showing that all transfected (withPNC-2-expressing plasmid) TUC-3 cells revert two weeks aftertransfection and selection of viable cells.

FIG. 3C is a photomicrograph of TUC-3 cells transfected withPNC-7-expressing plasmid, two weeks after transfection, showing cell andnuclear enlargement. These cells grow sluggishly into stable monolayers.

FIG. 4A is a photograph of gel blots showing co-immunoprecipitation ofjun-N-terminal kinase (JNK) (lane 6) and MAP kinase (MAPK or ERK) withHa-ras-p21, immunoprecipitated from oocytes that were induced tomaturity by microinjection of oncogenic Val 12-Ha-ras-p21 and blotted.

FIG. 4B is a photograph of gel blots showing co-immunoprecipitation ofjun-N-terminal kinase (JNK) (lane 6) and MAP kinase (MAPK or ERK) withHa-ras-p21, immunoprecipitated from oocytes that were induced tomaturity with insulin, which activates wild-type rs-p21. Only raf wasfound to immunoprecipitate with Ha-ras in these oocytes.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been surprisinglydiscovered that peptides designed from molecular modeling studies of theras-p21 protein induce phenotype reversion of a pancreatic carcinomacell line but have no effect on normal pancreatic acinar cell growth.The two peptides, designated PNC-2 and PNC-7, block oncogenicras-induced oocyte maturation but do not block insulin-activated wildtype ras-induced maturation.

Since various cancers involve expression of Val 12-p21 protein,inhibition of this protein as well as phenotypic reversion of cancerouscells expressing this protein upon treatment of PNC-2 and/or PNC-7,represents a valuable cancer therapy. One out of every three solidtumors involves expression of Val 12-p21. For example, between 50-75% ofcolon cancers, greater than 90% of pancreatic cancers, one third of allnon-small cell carcinomas of the lung, one fifth of gastric and bladdercancers, as well as many mesotheliomas involve expression of oncogenicras-p21 protein.

In accordance with the present invention, the peptides PNC-2, PNC-7,analogs and derivatives of such peptides, pharmaceutical preparationsand methods of treatment using PNC-2, PNC-7 peptides, analogs,derivatives thereof and pharmaceutical preparations based thereon, areuseful in treating a variety cancers. Preferably, the cancers which aretreated with the peptides pharmaceutical compositions and methods of thepresent invention are ras-induced cancers. Treatment of ras-inducedtumors by the compositions of the present invention convert malignantmasses into benign ones, allowing for the stopping of metastaticdisease.

In one aspect of the invention, there is provided a peptide comprisingat least about ten contiguous amino acids of the amino acid sequence:YREQIKRVKDSDDVP (SEQ ID NO: 1) or an analog or derivative thereof. In apreferred embodiment of the invention, the peptide is designated PNC-2and comprises the 15 amino acids as set forth in SEQ ID NO: 1.

In another aspect of the invention, there is provided a peptidecomprising at least about ten contiguous amino acids of the amino acidsequence: TIEDSYRKQVVID (SEQ ID NO:2), or an analog or derivativethereof. In a preferred embodiment of the invention, the peptide isdesignated PNC-7 and comprises the 13 amino acids as set forth in SEQ IDNO:2.

Preferably, the peptides having the amino acid sequence set forth in SEQID NO: 1 or SEQ ID NO:2, or an analog or derivative thereof, are fusedto a membrane-penetrating leader sequence. In order to be transportedacross a cell membrane and effect reversion of cancerous cells to normalphenotype, the leader sequence is preferably positioned at the carboxylterminal end of the peptide, analog, or derivative thereof. Preferably,the leader sequence comprises predominantly positively charged aminoacid residues. Examples of leader sequences which may be used inaccordance with the present invention include but are not limited topenetratin, Argx, TAT of HIV1, D-TAT, R-TAT, SV40-NLS,nucleoplasmin-NLS, HIV REV (34-50), FHV coat (35-49), BMV GAG (7-25),HTLV-II REX (4-16), CCMV GAG (7-25), P22N (14-30), Lambda N (1-22).Delta N (12-29), yeast PRP6, human U2AF, human C-FOS (139-164), humanC-JUN (252-279), yeast GCN4, and p-vec. Preferably, the leader sequenceis the penetratin sequence from antennapedia protein having the aminoacid sequence KKWKMRRNQFWVKVQRG (SEQ ID NO:3).

Pharmaceutical compositions comprising at least one of the subjectpeptides admixed with a pharmaceutically acceptable carrier are alsoprovided. In addition, methods for treating neoplastic disease (cancer)in a subject i.e., inducing phenotypic reversion of cancerous cells tobenign cells in a subject suffering from cancer, are provided. In oneembodiment, the method comprises administering to the subject, atherapeutically effective amount of a peptide comprising at least aboutten contiguous amino acids of the amino acid sequence: YREQIKRVKDSDDVP(SEQ ID NO: 1), or an analog or derivative thereof. Preferably, thepeptide or analog or derivative thereof is fused to amembrane-penetrating leader sequence and confers a normal phenotype oncancerous cells. Even more preferably, the membrane-penetrating leadersequence is fused to the carboxy terminal end of the peptide, analog, orderivative thereof. The cancer is preferably a ras-induced cancer.

In another embodiment, the method comprises administering to the subjectsuffering from cancer, a therapeutically effective amount of a peptidehaving the sequence set forth in TIEDSYRKQVVID (SEQ ID NO:2), or ananalog or derivative thereof. Preferably, the peptide or analog orderivative thereof is fused to a membrane-penetrating leader sequenceand confers a normal phenotype on cancerous cells. Even more preferably,the membrane-penetrating leader sequence is fused to the carboxyterminal end of the peptide, analog, or derivative thereof. The canceris preferably a ras-induced cancer.

In still another embodiment of the invention, the method comprisesadministering to a subject suffering from cancer, a therapeuticallyeffective amount of a mixture of peptides having the sequence set forthin SEQ ID NO: 1 and SEQ ID NO: 2, or analogs or derivatives thereof.Preferably, the peptides or analogs or derivatives thereof are fused toa membrane-penetrating leader sequence and confer a normal phenotype oncancerous cells. Even more preferably, the membrane-penetrating leadersequence is fused to the carboxy terminal end of the peptides, analogs,or derivatives thereof. The cancer is preferably a ras-induced cancer.

Leader sequences which function to import the peptides of the inventioninto a cell may be derived from a variety of sources. Preferably, theleader sequence comprises predominantly positively charged amino acidresidues since a positively charged leader sequence stabilizes the alphahelix of a subject peptide. Examples of leader sequences which may belinked to the peptides of the present invention are described in Futaki.S. et al (2001) Arginine-Rich Peptides, J. Biol. Chem. 276:5836-5840,and include but are not limited to the following membrane-penetratingleader sequences (numbering of the amino acid residues making up theleader sequence of the protein is indicated parenthetically immediatelyafter the name of the protein in many cases):

(SEQ ID NO: 3) penetratin (KKWKMRRNQFWVKVQRG); (SEQ ID NO: 27)(Arg)₈ or any poly-R from (R)₄-(R)₁₆; (SEQ ID NO: 4)HIV-1 TAT(47-60)(YGRKKRRQRRRPPQ) (SEQ ID NO: 5) D-TAT (GRKKRRQRRRPPQ);(SEQ ID NO: 6) R-TAT G(R)₉PPQ; (SEQ ID NO: 7) SV40-NLS (PKKKRKV);(SEQ ID NO: 8) nucleoplasmin-NLS (KRPAAIKKAGQAKKKK); (SEQ ID NO: 9)HIV REV (34-50)-(TRQARRNRRRRWRERQR); (SEQ ID NO: 10)FHV (35-49) coat (RRRRNRTRRNRRRVR); (SEQ ID NO: 11)BMV GAG (7-25)-(KMTRAQRRAAARRNRWTAR); (SEQ ID NO: 12)HTLV-II REX 4-16-(TRRQRTRRARRNR); (SEQ ID NO: 13)CCMV GAG (7-25)-(KLTRAQRRAAARKNKRNTR); (SEQ ID NO: 14)P22 N (14-30)(NAKTRRHERRRKLAIER); (SEQ ID NO: 15)LAMBDA N(1-22)(MDAQTRRRERRAEKQAQWKAAN); (SEQ ID NO: 16)Phi N (12-29)(TAKTRYKARRAELIAERR); (SEQ ID NO: 17)YEAST PRP6 (129-124)(TRRNKRNRIQEQLNRK); (SEQ ID NO: 18)HUMAN U2AF (SQMTRQARRLYV); (SEQ ID NO: 19)HUMAN C-FOS (139-164) KRRIRRERNKMAAAKSRNRRRELTDT; HUMAN C-JUN (252-279)(SEQ ID NO: 20) (RIKAERKRMRNRIAASKSRKRKLERIAR); (SEQ ID NO: 21)YEAST GCN4 (KRARNTEAARRSRARKLQRNIKQ); (SEQ ID NO: 22)KLALKLALKALKAALKLA; (SEQ ID NO: 23) p-vec LLIILRRRIRKQAKAHSK.

Other membrane penetrating leader sequences may also be used. Suchsequences are widely available and are described e.g., in Scheller ctal. (2000) Eur. J. Biochem. 267:6043-6049, and Elmquist et al., (2001)Exp. Cell Res. 269:237-244.

Preferably, the positively charged leader sequence of the penetratinleader sequence of antennapedia protein is used. This leader sequencehas the following amino acid sequence: KKWKMRRNQFWVKVQRG (SEQ ID NO: 3).Preferably, the leader sequence is attached to the carboxyl terminal endof a subject peptide to enable the synthetic peptide to effectphenotypic reversion of cancerous cells.

Structurally related amino acid sequences may be substituted for thedisclosed sequences set forth in SEQ ID NOs: 1 or 2 in practicing thepresent invention. Amino acid insertional derivatives of the peptides ofthe present invention include amino and/or carboxyl terminal fusions aswell as intra-sequence insertions of single or multiple amino acids.Insertional amino acid sequence variants are those in which one or moreamino acid residues are introduced into a predetermined site in asubject peptide although random insertion is also possible with suitablescreening of the resulting product. Deletional variants may be made byremoving one or more amino acids from the sequence of a subject peptide.Substitutional amino acid variants are those in which at least oneresidue in the sequence has been removed and a different residueinserted in its place. Typical substitutions are those made inaccordance with the following Table 1:

TABLE I Suitable residues for amino acid substitutions Original ResidueExemplary Substitutions Ala (A) Ser Arg (R) Lys Asn (N) Gln; His Asp (D)Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Pro His (H) Asn, Gln Ile(I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Ile Phe(F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; PheVal (V) Ile; Leu

When the synthetic peptide is derivatised by amino acid substitution,the amino acids are generally replaced by other amino acids having likeproperties such as hydrophobicity, hydrophilicity, electronegativity,bulky side chains and the like. As used herein, the terms “derivative”,“analogue”, “fragment”, “portion” and “like molecule” refer to a subjectpeptide having the amino acid sequence as set forth in SEQ ID NOs: 1 or2, having an amino acid substitution, insertion, addition, or deletion,as long as said derivative, analogue, fragment, portion, or likemolecule retains the ability to enter and effect phenotypic reversion ofcancer cells, while having no effect on normal, non-cancerous cells.

The synthetic peptides of the present invention may be synthesized by anumber of known techniques. For example, the peptides may be preparedusing the solid-phase technique initially described by Merrifield (1963)in J. Am. Chem. Soc. 85:2149-2154. Other peptide synthesis techniquesmay be found in M. Bodanszky et al. Peptide Synthesis, John Wiley andSons, 2d Ed., (1976) and other references readily available to thoseskilled in the art. A summary of polypeptide synthesis techniques may befound in J. Sturart and J. S. Young, Solid Phase Peptide Synthesis,Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also besynthesized by solution methods as described in The Proteins. Vol. II,3d Ed., Neurath, H. et al., Eds., pp. 105-237. Academic Press, New York,N.Y. (1976). Appropriate protective groups for use in different peptidesyntheses are described in the texts listed above as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York,N.Y. (1973). The peptides of the present invention may also be preparedby chemical or enzymatic cleavage from larger portions of the ras-p21protein or from the full-length ras-p21 protein. Likewise, leadersequences for use in the synthetic peptides of the present invention maybe prepared by chemical synthesis or enzymatic cleavage from largerportions or the full-length proteins from which such leader sequencesare derived.

Additionally, the peptides of the present invention may also be preparedby recombinant DNA techniques. For most amino acids used to buildproteins, more than one coding nucleotide triplet (codon) can code for aparticular amino acid residue. This property of the genetic code isknown as redundancy. Therefore, a number of different nucleotidesequences may code for a particular subject peptide. The presentinvention also contemplates use of a deoxyribonucleic acid (DNA)molecule that defines a gene coding for, i.e., capable of expressing asubject peptide or a chimeric peptide from which a peptide of thepresent invention may be enzymatically or chemically cleaved.

Consistent with the observed properties of the peptides of theinvention, the subject peptides may be used to induce phenotypicreversion of neoplastic or malignant cells, i.e., cancer cells inanimals, preferentially humans. The synthetic peptides of the presentinvention are thus administered in an effective amount to convertmalignant cells or masses into benign cells or masses in a subjectanimal or human. Reversion of cancerous cells or masses into benigncells or masses would have an additional benefit of halting metastasisand the spread of metastatic disease.

The synthetic peptides of the present invention may be administeredpreferably to a human patient as a pharmaceutical composition containinga therapeutically effective dose of at least one synthetic peptideaccording to the present invention together with a pharmaceuticalacceptable carrier. The term “therapeutically effective amount” or“pharmaceutically effective amount” means the dose needed to produce inan individual, phenotypic reversion of neoplastic or malignant cells,i.e., cancer cells to benign or non-cancerous cells.

Preferably, compositions containing one or more of the syntheticpeptides of the present invention are administered intravenously for thepurpose of treating neoplastic or malignant disease such as cancer.Examples of different cancers which may be effectively treated using oneor more the peptides of the present invention include but are notlimited to: breast cancer, prostate cancer, lung cancer, cervicalcancer, colon cancer, melanoma, pancreatic cancer and all solid tissuetumors (epithelial cell tumors) and cancers of the blood including butnot limited to lymphomas and leukemias. Preferably, the cancer to betreated in accordance with the present invention is a ras-induced cancersuch as colon cancer, pancreatic cancer, non-small cell carcinoma of thelung, gastric cancer, bladder cancer and mesotheliomas. Most preferablythe cancer to be treated is pancreatic cancer.

Administration of the synthetic peptides of the present invention may beby oral, intravenous, intranasal, suppository, intraperitoneal,intramuscular, intradermal or subcutaneous administration or by infusionor implantation. When administered in such manner, the syntheticpeptides of the present invention may be combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of the other ingredients, except that they must bepharmaceutically acceptable, efficacious for their intendedadministration, cannot degrade the activity of the active ingredients ofthe compositions, and cannot impede importation of a subject peptideinto a cell. The peptide compositions may also be impregnated intotransdermal patches, or contained in subcutaneous inserts, preferably ina liquid or semi-liquid form which patch or insert time-releasestherapeutically effective amounts of one or more of the subjectsynthetic peptides.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. The ultimatesolution form in all cases must be sterile and fluid. Typical carriersinclude a solvent or dispersion medium containing, e.g., water bufferedaqueous solutions, i.e., biocompatible buffers, ethanol, polyols such asglycerol, propylene glycol, polyethylene glycol, suitable mixturesthereof, surfactants or vegetable oils. Sterilization may beaccomplished utilizing any art-recognized technique, including but notlimited to filtration or addition of antibacterial or antifungal agents.Examples of such agents include paraben, chlorbutanol, phenol, sorbicacid or thimerosal. Isotonic agents such as sugars or sodium chloridemay also be incorporated into the subject compositions.

As used herein, a “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic agents and the like. The use of such media and agentsare well known in the art.

Production of sterile injectable solutions containing the subjectsynthetic peptides is accomplished by incorporating one or more of thesubject synthetic peptides described hereinabove in the required amountin the appropriate solvent with one or more of the various ingredientsenumerated above, as required, followed by sterilization, preferablyfilter sterilization. In order to obtain a sterile powder, the abovesolutions are vacuum-dried or freeze-dried as necessary.

Inert diluents and/or assimilable edible carriers and the like may bepart of the pharmaceutical compositions when the peptides areadministered orally. The pharmaceutical compositions may be in hard orsoft shell gelatin capsules, be compressed into tablets, or may be in anelixir, suspension, syrup or the like.

The subject synthetic peptides are thus compounded for convenient andeffective administration in pharmaceutically effective amounts with asuitable pharmaceutically acceptable carrier in a therapeuticallyeffective dosage. Examples of a pharmaceutically effective amountinclude peptide concentrations in the range from about at least about 25ug/ml to at least about 300 ug/ml.

A precise therapeutically effective amount of synthetic peptide to beused in the methods of the invention applied to humans cannot be stateddue to variations in stage of neoplastic disease, tumor size andaggressiveness, the presence or extent of metastasis, etc. In addition,an individual's weight, gender, and overall health must be consideredand will affect dosage. It can be generally stated, however, that thesynthetic peptides of the present invention be administered in an amountof at least about 10 mg per dose, more preferably in an amount up toabout 1000 mg per dose. Since the peptide compositions of the presentinvention will eventually be cleared from the bloodstream,re-administration of the pharmaceutical compositions is indicated andpreferred.

The synthetic peptides of the present invention may be administered in amanner compatible with the dosage formulation and in such an amount aswill be therapeutically effective. Systemic dosages depend on the age,weight, and condition of the patient and the administration route. Anexemplary suitable dose for the administration to adult humans rangesfrom about 0.1 to about 20 mg per kilogram of body weight. Preferably,the dose is from about 0.1 to about 10 mg per kilogram of body weight.

In accordance with the present invention, there is also provided amethod of treating neoplastic disease. The method comprisesadministering to a subject in need of such treatment, a therapeuticallyeffective amount of a synthetic peptide hereinbefore described,including analogs and derivatives thereof. Thus for example, in oneembodiment, an effective amount of a peptide comprising at least aboutten contiguous amino acids as set forth in SEQ ID NO: 1 or an analog orderivative thereof, fused on its carboxy terminal end to a leadersequence may be administered to a subject. An effective amount of apeptide having the amino acid sequence as set forth in SEQ ID NO:2 or ananalog or derivative thereof, fused on its carboxy terminal end to aleader sequence may also be administered to a subject. In accordancewith a method of treatment, a mixture of synthetic peptides may beadministered. Thus, for example, in addition to administering one of thepeptides, or analogs or derivatives thereof hereinbefore described in aneffective amount, mixtures of two or more peptides or analogs orderivatives hereinbefore described may be administered to a subject.

In another aspect of the present invention, there are providedexpression vehicles comprising replication incompetent Adenovirus (AdV)and having a promoter sequence operably linked to a coding sequence fora subject peptide, e.g., nucleotide sequences encoding those peptidesdescribed above i.e., SEQ ID NO: 1, SEQ ID NO: 2, or analogs orderivatives thereof as described fully above. As described above, morethan one triplet (codon) can code for a particular amino acid residue.Table 2 shows the different combinations of codons which may be used toencode the amino acid sequence set forth in SEQ ID NO: 1. Table 3 showsthe different combinations of codons which may be used to encode theamino acid sequence set forth in SEQ ID NO: 2. The amino acid sequenceof SEQ ID NO: 1 I is shown in the top line of Table 2 in bold. The aminoacid sequence of SEQ ID NO: 2 is shown in the top line of Table 3 inbold.

TABLE 2 Y R E Q I K R V K D S D D V P TAT AGA GAA CAA ATT AAG CGT GTTAAG GAT TCT GAT GAT GTT CCU TAC AGG GAG CAG ATC AAA CGC GTC AAA GAC TCCGAC GAC GTC CCC CGT ATA CGA GTA TCA GTA CCA CGC CGG TCG CCG CGA AGA AGTCGG AGG AGC

TABLE 3 T I E D S Y R K Q V V I D ACT ATT GAA GAT TCT TAT CGT AAG CAAGTT GTT ATT GAT ACC ATC GAG GAC TCC TAC CGC AAA CAG GTC GTC ATC GAC ACAATA TCA CGA GTA GTA ATA ACG TCG CGG AGT AGA AGC AGG

With respect to using nucleotide sequences encoding an analog orderivative of the amino acid sequences set forth in SEQ ID NOs: 1 or 2,one skilled in the art can refer to a table of the Genetic Code toselect appropriate codons.

A number of different classes of Ad vectors exist, and may be used inthe methods of the present invention. Such Ad vectors are described inthe literature and are readily available. See refs. 26 and 27. Forexample, in accordance with the present invention, an Ad vector may beused wherein the E1 and/or E3 genes have been removed, allowing theintroduction of up to about 6.5 kb of transgene under the control of aheterologous promoter. See ref. 28. The defective E1 viruses may bepropagated in an E1-complementing cell line, such as 293A cells, whichcells provided the E1 gene in trans.

Alternatively, an Ad vector may be used which in addition to lacking theE1 and E3 genes, also lack the E2 genes. See e.g., refs. 29 and 30.

In addition, helper-dependent (HD) or gutted vectors deleted of most orall Ad coding sequences may be used in accordance with the presentinvention. Such vectors have great potential as gene transfer vectorsfor gene therapy since long term expression of therapeutic genes havebeen observed in mice as well as monkeys. The production of these guttedvectors in tissue culture requires a complementing helper virus toprovide the proteins required for growth and assembly of the guttedvector in trans. See refs. 31-33. The disclosures of these papers andall references cited herein, are incorporated by reference as if fullyset forth.

As discussed above, in the present application directed to viral therapyof neoplastic disease, e.g., cancer, where the goal of the therapy isclearance of the target tissue, a host anti-Ad immune response targetingthe vector infected cells is considered desirable. Thus, a gutted Advector may not be as preferred as some of the earlier generation vectorswhich elicit a stronger immune response in the host.

An Ad vector may be based on a two-plasmid system, an entry plasmid anda destination vector made from E1 and E3 gene deleted adenoviral genomethat contains a promoter operably linked to a nucleotide sequenceencoding one of the peptides described above (SEQ ID NOs: 1 or 2) aswell as analogs or derivatives thereof. The two-plasmid system isthoroughly described in refs. 28, 34, and 35. The E1 and E3 genedeletions prevent the virus from replicating in cells that do notexpress E1 and E3 proteins.

For example, the entry plasmid contains the gene encoding a subjectpeptide which plasmid is cloned into the AdV via a lambda recombinationreaction. The replication incompetent vector may be propagated in 293Acells, which are bioengineered human embryonic kidney cells transformedby AdV genomic DNA (Wang et al., 2000). This cell line supplements thedeficient genes required for viral replication.

The replication incompetent AdV vectors of the present invention can beconstructed using standard recombinant DNA methods. Standard techniquesfor the construction of vectors are well-known to those of ordinaryskill in the art and can be found in references such as Sambrook,Fritsch and Maniatis, 1989, or any of a number of laboratory manuals onrecombinant DNA technology that are widely available. A variety ofstrategies are available for ligating fragments of DNA, the choice ofwhich depends on the nature of the termini of the DNA fragments and canbe readily determined by the skilled artisan. There are a number ofdifferent promoters which may be operably linked to the nucleotidesequences encoding a subject peptide.

The promoter should function in the cells of a subject undergoing viraltherapy with a subject AdV vector. There are a number of widelyavailable promoters which may be used in the AdV vectors of the presentinvention. Examples of such promoters include, but are not limited to:CMV, SV40, RSV, LTR, beta-actin, EF-1 alpha, Gal-E1b, UbC, beta-Casein,EM-7, EF, TEF1, CMV-2 and Bsd. In a preferred embodiment, the promoteris CMV.

The recombinant vectors may then be subsequently rebuilt into intactviruses using standard methods such as that described in ref. 36, whichis incorporated by reference herein as if fully set forth. Otherreferences which describe rebuilding recombinant vectors into intactviruses include ref. 37, also incorporated by reference herein as iffully set forth.

Once a subject AdV vector is constructed, it may be used to treatpatients suffering from different types of cancer. Therapy of neoplasticdisease (cancer) may be accomplished by administering to a patientsuffering from such disease a composition comprising the adenovirusvectors of the present invention. A human patient or nonhuman mammalsuffering from a carcinoma may be treated by administering an effectiveantineoplastic dosage of a subject vector. The subject AdV vectorscomprising a promoter operably linked to a nucleotide encoding a subjectpeptide are useful in treating a number of different cancers includingbut not limited to breast cancer, prostate cancer, lung cancer, cervicalcancer, colon cancer, melanoma, pancreatic cancer, all solid tissuetumors (epithelial cell tumors) and cancers of the blood including butnot limited to lymphomas and leukemias. In a preferred embodiment, thecancer to be treated is pancreatic cancer.

Suspensions of infectious adenovirus particles may be applied toneoplastic tissue by various routes, including intravenous,intraperitoneal, intramuscular, subdermal, and topical. Other routesinclude inhalation as a mist (e.g., in treating lung cancer) or directapplication such as by swabbing a tumor site, e.g., cervical carcinoma,or during surgery if necessary. An adenovirus suspension may also beadministered by infusion, e.g., into the peritoneal cavity for treatingovarian cancer. Other suitable routes include direct injection into atumor mass, such as a breast tumor, via enema (colon cancer) or catheterin the case of bladder cancer.

The actual dosage may vary from patient to patient based on the age,weight, type and progression of cancer, location of tumor(s), presenceof metastases, and overall condition of the patient. It can generally besaid, however, that an adenovirus suspension containing about 10³ toabout 10¹⁵ or more virion particles per ml may be administered.Re-administration of the AdV vector suspension may be performed asnecessary.

The AdV vectors of the present invention may be admixed in a sterilecomposition containing a pharmacologically effective dosage of one ormore subject AdV vectors. Generally speaking, the composition willcomprise about 10³ to about 10¹⁵ or more AdV particles in an aqueoussuspension. The sterile composition is usually an aqueous solution suchas e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like.Such compositions may contain pharmaceutically acceptable auxiliarysubstances e.g., to mimic physiological conditions such as pH adjustingand buffering agents, toxicity adjusting agents and the like, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate, etc. The compositions may also comprise excipients thatenhance infection of cells by the subject AdV vectors.

The following examples further illustrate the invention, and are notmeant in any way to limit the scope thereof:

Example I Materials and Methods

Peptides.

Three peptides, attached to the penetratin leader sequence,KKWKMRRNQFWVKVQRG (SEQ ID NO:3), designated as “leader,” on theircarboxyl terminal ends, were synthesized by solid phase methods: the tworas-p21 peptides corresponding to p21 residues 35-47 (TIEDSYRKQVVID)(SEQ ID NO:2) and 96-110 (YREQIKRVKDSDVP) (SEQ ID NO: 1), denoted asPNC-7 and PNC-2, respectively; and the negative control X13 sequence(from mammalian cytochrome P450) (MPFSTGKRIMLGE) (SEQ ID NO:28). Withthe penetratin sequence attached to their carboxyl terminal ends, eachof these peptides is denoted as PNC-7-leader, PNC-2-leader andX13-leader, respectively. All peptides were purified to >95 percentpurity.

Plasmids. Construction of the plasmids that express the Ha-ras Val12-p21 peptide sequence 96-110 (PNC2) and the control X13 peptide frommammalian cytochrome p450 has been described (21). The nucleotidesequences for PNC-2 and X13 peptides are given in ref. 21. Thenucleotide sequences, including the 5′ sticky end, used to encode thePNC-7 peptide were 5′-T CGA GCC ACC ATG GGG ACC GAG GAT TCT TAC AGA AAACAA GTG GTT ATA GAT TAA C (SEQ ID NO: 24) and 3′-CGG TGG TAC CCC TGG TATCTC CTA AGA ATG TCT TTT GTT CAC CAA TAT CTA ATT GGG CC (SEQ ID NO:25).Briefly, all of the oligonucleotides (plus and minus strands) encodingeach sequence (PNC-2, PNC-7 and X13) and including a Not1(5′) andKpn1(3′) restriction site were synthesized by solid phase methods;sequential degradation of each oligonucleotide confirmed its sequence.These oligonucleotides were then incorporated into the pOPRSVI/MCSvector from the Lac switch II isopropylthioglucose (IPTG)-induciblemammalian expression system from Stratagene (LaJolla, Calif.) by cuttingthis vector with Kpn1 and Not1 and then ligating the oligomers into theplasmid with T4 ligase overnight at 4° C. The vectors containing thecloned oligonucleotides were transfected into DH5á competent cells(Gibco-BRL, Grand Island, N.Y.) and spread on LBamp plates for overnightincubation. Colonies from each plate were selected and grown at 37° C.in 5 ml of LBamp liquid media. DNA was prepared by the Qiagen (Valencia,Calif.) miniprep procedure, cut with Kpn1/Not1, and run on 2 percentagarose/TAE to estimate the size of the inserts. Clones with the correctsize DNA inserts were regrown in 500 ml LBamp overnight at 37° C., andplasmids were then purified by the Qiagen maxiprep method. An aliquot ofeach positive DNA was sequenced using T3 or T7 primers.

We note that, in our former paper describing these plasmids, an erroroccurred in the 5′ nucleotide sequence encoding PNC-2. This sequenceshould have read:

Upper (SEQ ID NO: 26) 5′-CGCCGCCATGGGCTACAGGGAGCAGATCAAGAGGGTGAAGGACAGCGACCACGTGCCCTA

In our original paper the highlighted C was inadvertently omitted.

Cells.

As described in several prior publications (16,17,20), we have developedtwo cell lines, one a normal contact-inhibited line of rat pancreaticacinar cells, called BMRPA1.430 (BMRPA1) cells and the other apancreatic acinar carcinoma obtained by transfection of BMRPA1 cellswith a plasmid containing an activated human K-ras oncogene [single basemutation at codon 12, valine substitution for the wild type glycine inthe ras protein (K-ras^(val12)); a kind gift of Dr. M. Perucho (CIBR, LaJolla, Calif.)] and a neomycin resistance gene. BMRPA1 cells have anepithelial cell phenotype, form acinar structures in culture, have noc-ki-ras nor p53 mutations, are unable to grow in anchorage-independentconditions and do not form tumors in Nu/Nu mice (17). In addition, theyphenotypically maintain differentiated cell functions such as continuedenzyme production and activation of zymogen secretion by secretagogue.On the other hand, ras-transformed BMRPA1 or TUC-3 cells, selected aftertransfection for their basis resistance to G418 and the overexpressionof K-ras^(val12), no longer display an epithelial cell phenotype andacinar cell functions; they grow significantly faster than BMRPA1 cells,have a transformed spindle cell phenotype and form colonies underanchorage-independent conditions in vitro and tumors in vivo in nudemice.

Peptide Incubation Experiments.

Approximately 300,000 cells (either BMRPA1 or TUC-3) were plated in eachof six wells and were allowed to adhere overnight. In one set ofexperiments, the initial media consisted of DMEM with 10% fetal bovineserum that contained no peptide. In another set of experiments, theinitial media contained peptide. In the first set, media containingpeptide was added after 24 hours; in both sets, after the first 24hours, the media was changed every 24 hours and always contained peptideat a particular concentration. Cells were observed daily for three weeksfor changes in morphology and growth characteristics. Peptides werepresent at concentrations of 1, 10, 50, 100 and 100 ug/ml.

Transfection Experiments.

Approximately 300,000 TUC-3 cells were plated overnight in a six-welldish and were allowed to adhere overnight. To three wells, 5.5 ug ofeither PNC-2 or PNC-7 plasmid were added and, to the other three wells,5.6 ug of X13 plasmid were added. To each of these wells. Superfecttransfection agent (Qiagen) was added, using the Qiagen protocol, toenhance transfection efficiencies. We found that a 1:2 ratio of plasmidDNA to Superfect reagent gave the highest transfection efficiencies whencompared with 1:5 and 1:10 ratios. Treated cells were then plated inselective medium containing 100 ug/ml G418 and 200 ug/ml of ampicillintogether with 1 mM isopropylthioglucose (IPTG). The cells were washedand the medium changed every 24 hours. Viable cells were observed formorphology and growth characteristics over a two-week period.

Explantation of Cells into Nude Mice.

To evaluate cells that appeared to be morphologically revertant to thenormal phenotype, approximately 5×10 morphologically revertant TUC-3cells treated for two weeks with 100 ug/ml of PNC-2 were injectedsubcutaneously into the posterior cervical fatpad of each of five Nu/Numice. Similarly, 5×10 untreated TUC-3 cells were explanted into anotherfive Nu/Nu mice. Daily observations, over a 120 day period, were made onboth sets of mice to determine if tumor nodules appeared at the site ofinjection.

Example II Results

Effects of Peptides on TUC-3 and BMRPA1 Cells.

FIGS. 1A and 1C show the morphology of untreated TUC-3 pancreaticcarcinoma cells and their normal counterpart BMRPA1 pancreatic acinarcells, respectively. The former are not-contact-inhibited and do notform monolayers but are “heaped up” on one another with considerablepleomorphism between cells and indistinct cell boundaries. The latterform contact-inhibited monolayers with well-defined cell boundaries.Panel B in FIG. 1 shows that incubation of the X13-leader controlpeptide with TUC-3 cells for two weeks has no effect on theirtransformed morphologies. As expected, incubation of this controlpeptide with BMRPA1 cells has no effect (not shown). Incubation ofBMRPA1 cells with PNC2-leader peptide likewise has no effect on themorphology of these cells (Panel D in FIG. 1).

Effects of PNC-2-Leader and PNC-7-Leader on TUC-3 Cells.

Treatment of TUC-3 cells with PNC-2-Leader (100 ug/ml) for 1 weekresults in a change in cell morphology as shown in FIG. 2A. As can beseen in this figure, the cells appear very similar to BMRPA1 cells (FIG.1C); the cells grow into contact-inhibited monolayers and show distinctcell boundaries. This effect was achieved at concentrations as low as 1ug/ml. At this low concentration, complete phenotypic reversion wasachieved after two weeks. After one day of treatment, foci of acinarcellular differentiation appear; an example of a focus of revertantcells is shown in FIG. 2B.

Treatment of TUC-3 cells with PNC-7-leader peptide at concentrations of100 and 200 ug/ml likewise resulted in phenotypic reversion of the cellsas shown in FIG. 2C for cells growing into confluence. In contrast tothe results obtained with PNC-2-leader peptide, complete reversion aftertwo weeks of incubation of TUC-3 cells with PNC-7-leader was achievedonly at concentrations $100 ug/ml.

Transfection of TUC-3 Cells with Inducible Plasmids Encoding PNC-2 andX13 Peptides.

Since both PNC-2- and 7-leader peptides induce phenotypic reversionwhile X13-leader control peptide does not, we conclude that induction ofreversion is specific to the two ras-p21 peptides and that the leadersequence, besides enabling membrane penetration, does not contribute tothe induction of phenotypic reversion. To test the latter conclusionfurther, i.e. that PNC-2 and PNC-7 peptides alone, without the leadersequence, can induce phenotypic reversion, we prepared plasmids encodingthese and the negative control X13 sequences and transfected them intoTUC-3 cells. In a previous publication, we described the preparation ofthese plasmids which simultaneously confer G418 and ampicillinresistance under the lac promoter (21). We co-microinjected theseplasmids with Val 12-p21 protein into Xenopus laevis oocytes and foundthat oocytes injected with either PNC-2 or PNC-7 but not X13 plasmid, inthe presence of isopropylthioglucose (IPTG), did not undergo maturation(21). When we transfected each of these plasmids into TUC-3 cellsgrowing in the selective medium, viable cells expressing X13 peptidecontinued to grow in the presence of IPTG and exhibited the transformedmorphology shown in Panel A in FIG. 1.

On the other hand, during a period of two weeks post-transfection withPNC-2 plasmid, all viable TUC-3 cells became progressivelydifferentiated as shown in panels A (after 1 week) and B (after 2 weeks)of FIG. 3. As can be seen in panel A of FIG. 3, after one week, manycells adopted the untransformed phenotype (center and left of panel A)while some cells exhibited the transformed phenotype (right side offigure). At the end of two weeks, all cells exhibited the morphologyshown in panel B of FIG. 3. As can be seen in this figure, the cellshave distinct cell boundaries and exhibit the same morphology asuntransformed BMRPA1 cells in growth phase. These cells eventually grewinto contact-inhibited monolayers with a morphology that was the same asshown in FIG. 1, panel C.

Transfection of TUC-3 cells with PNC-7 plasmid exhibited the phenotypeshown in Panel C of FIG. 3. These cells, which are seen to be enlargedwith enlarged nuclei but have distinct cell boundaries, grew onlysluggishly to confluence, and strongly resemble viable revertant cellsthat resulted from the treatment of TUC-3 cells with the anti-proteinkinase C inhibitor, CGP 41 251 (16). These cells fail to grow in softagar (16).

Morphologically Revertant Cells do not Form Tumors in Nude Mice.

To test whether morphologically revertant cells were functionallyrevertant, 5×10⁶ cells treated for two weeks with 100 ug/ml PNC-2-leaderpeptide were explanted subcutaneously into each of five nude mice whilethe same number of untreated TUC-3 cells were concomitantly similarlyexplanted. The results, shown in Table 4, indicate that morphologicallyrevertant cells fail to form tumors up to two months after reversionwhile untreated cells form tumors rapidly (within 1 week). At threeweeks, all of the nude mice injected with untreated TUC-3 cells werefound to have large primary nodules and multiple other nodules andmetastatic cancer, with ascites and other sites. Similar results (notshown) to those obtained with PNC-2-leader peptide-treated TUC-3 cellswere obtained for morphologically revertant cells resulting from TUC-3cells treated with PNC-7-leader peptide.

Both PNC-2 and PNC-7 peptides block mitogenic signaling by oncogenicras-p21 in oocytes but have little effect on signaling byinsulin-activated wild-type cellular p²¹ (5). This finding suggested tous that growth of mammalian cells transformed by oncogenic ras-p21 canbe selectively blocked by these peptides without affecting normal growthprocesses.

Both PNC-2 and PNC-7 peptides induce 100 percent phenotypic reversion ofras-transformed pancreatic (TUC-3) cancer cells and have no apparenteffects on the growth of the normal counterpart BMRPA1 cell line. Thiseffect is specific since neither the X13-leader control peptide nor theplasmid encoding it has any effect on TUC-3 cell proliferation. That thePNC-2 and 7 sequences and not the leader sequence, are responsible forthis effect is supported by the absence of any effect on TUC-3 cells ofthe X13-leader peptide and by the finding that the plasmids encodingPNC-2 and PNC-7 without the leader sequence induces the same observedphenotypic reversion.

A surprising finding is that the phenotypic reversion induced by bothpeptides occurs over a prolonged period of time (120 days), as revealedby the absence of any tumor growth of these cells when explanted intonude mice. Since the half-lives of these peptides is expected to be muchshorter than two months, their effects are not likely to be caused bytheir continuing presence. Significantly, the prolonged reversion effectappears to be independent of the site of action of these peptides sincePNC-2 blocks oncogenic ras-p21-JNK interactions (5,11,12) while PNC-7blocks oncogenic ras-p21-raf interactions (14,15).

It is possible that both peptides activate rapid expression of otherproteins that interfere with oncogenic ras-induced cell proliferation.This type of effect has been observed in human pancreatic carcinomacells induced to revert by the agent azatyrosine that is known to induceexpression of the ras recision gene (rrg) (22,23) and which alsoselectively blocks oncogenic ras-p21-induced oocyte maturation (13).Another possibility is that each peptide, by blocking signaltransduction unique to the oncogenic ras-p21-induced pathway, allowsother inhibitory processes continuously to deactivate critical elementsin this pathway.

The activity of both PNC-2 and PNC-7 peptides contrasts with that ofanother oncogenic-ras-p21-specific inhibitor, the staurosporinederivative, CGP 41 251, that selectively inhibits protein kinase C(PKC)(24). This agent blocks oncogenic ras-p21-induced oocyte maturationbut has much less effect on insulin-activated wild-type ras-p21-inducedmaturation (13). In contrast to the results with PNC-2- and 7-leaderpeptides, this agent induces both necrosis and phenotypic reversion ofTUC-3 cells (16) and is cytotoxic to BMRPA1 cells, although survivingcells grow rapidly into stable monolayers (16). Cytotoxicity of CGP 41251 may be due to its blocking critical PKC-dependent cell processesthat may not be involved in cell proliferation.

In prior studies, it had been found that PKC and JNK require each otherspresence on the oncogenic ras-p21 signal transduction pathway (25). Inaddition, PNC-2 synergizes with CGP 41 251 in TUC-3 cells in that itsignificantly lowers its IC₅₀ for induction of cytotoxicity to a levelthat is not toxic to BMRPA1 cells (16). This finding suggests thepossibility that PNC-2, which blocks ras-p21-induced activation of JNK(5), inhibits the mutual PKC-JNK activation cycle thereby removing animportant activation process, resulting in facilitation of inhibition byCGP 41 251.

Evidently PNC-2 and PNC-7 exert a more selective effect that is specificto the oncogenic ras-p21 pathway, hence the lack of cytotoxicity ofthese peptides. This finding indicates that these peptides are useful inthe treatment of ras-induced human tumors.

TABLE 4 Growth of TUC-3 Cells and Morphologically Reverted TUC-3 CellsTreated with PNC-2 Peptide Explanted into Nude Mice.^(a) Tumor NoduleSize (mm)^(b) Time (days) TUC-3 Cells PNC-2-Treated TUC-3 0 0.0 0.0 7 4.8 ± 1.8 0.0 14 11.7 ± 2.3 0.0 21  14.8 ± 3.6^(c) 0.0 28 — 0.0 41 —0.0 56 — 0.0 ^(a)An amount of 5 × 10⁶ TUC-3 cells was injected into theposterior cervical fat pad of each of 5 nude mice, and the same numberof TUC-3 cells treated for 2 weeks with PNC-2-leader peptide wasinjected into the posterior cervical fat pad of another 5 nude mice.^(b)Expressed as the means ± SD for the five mice in each group.^(c)Multiple nodules and tumor metastasis with ascites occurred in allfive mice at this time. Further observations were thereforediscontinued.

Example III PNC-2 and PNC-7 Block the Interaction of JNK and MAP Kinasewith Val 12-Ras p21 Inside the Cell

In these experiments, the Ha-ras form of Val 12-p21 was injected intooocytes (100 ug/ml, 50 nl per oocyte) either alone or together withinhibitory p21 peptide (residues 96-110 shown in this figure). Matureoocytes (non-matured oocytes were used with inhibitory p21 96-110peptide since it strongly inhibits maturation) were collected after 24hours (about 50% maturation, approximately 100 oocytes) and subjected tolysis in buffer consisting of 0.35 M LiCl, 50 mM HEPES, pH 7.6, 1 mMEGTA, 1 mM dithiothreitol (DDT), 2 mM MgCl, 50 mM NPP, 1 mM sodiumvanadate, and an inhibitor ‘cocktail’ consisting of 1 ug/ml each of theprotease inhibitors: pepstatin, leupeptin and aprotinin; and thephosphatase inhibitors: 1 mM sodium orthovanadate and 5 mM sodiumfluoride). The lysate was centrifuged for 15 min at 17000×g at 4° C.,and the supernatant was either used directly. The lysates were thensubjected to immunoprecipitation using an anti-Ha-ras antibody(CalBiochem). In this procedure, cell lysate was first pre-cleared byincubation with 50 ul of protein A beads for 1 hr at room temperature,followed by centrifugation. Anti-Ha-ras antibody was added to the lysatesuch that 0.1 ug antibody was added per 250 ug of pre-cleared lysateprotein. A volume of 25 ul protein A agarose beads (Sigma) was thenadded to the incubation mixture, and the resulting mixture was incubatedovernight at 4° C., after which the mixture was centrifuged, and theimmunoprecipitate was washed three times with 0.5 ml of kinase buffer asdescribed above. Immunoprecipitates were subjected to SDS-PAGE asdescribed above in the preceding paragraph and blotted with anti-Ha-ras(1:2000 with 0.25% BSA), anti-raf (CalBiochem, San Diego, Calif.),diluted 1:2000 with 0.25% BSA, anti-JNK polyclonal antibody (1:2000),anti-MEK (CalBiochem) and anti-MAPK, diluted 1:2000 with 0.25% BSA. Allincubations were performed as described in the preceding paragraph,i.e., for 12 hr at 4° C., after which the membranes were washed threetimes with Tris-buffered saline with Triton (TBS-T) and incubated withanti-rabbit secondary antibody (Pierce, Rockford, Ill.) at 1:20000dilution. Detection was accomplished using the ECL chemiluminescencedetection kit (Pierce). An identical set of experiments was performedwith oocytes incubated for 24 h with 10 ug/ml insulin (Sigma, St. Louis,Mo.).

FIG. 4A shows the results of injected Val 12-p21 forming a complex withraf, MEK, JNK and MAPK (ERK). Oocytes that matured after being injectedwith Val 12-Ha-ras-p21 were lysed and immunoprecipitated withanti-Ha-ras antibody. The immunoprecipitate was blotted with anti-raf(lane 2), anti-MEK (lane 4), anti-JNK (lane 6) and anti-MAPK (lane 8).Oocytes were also injected with Val 12-p21 and ras-p21 inhibitorypeptide 96-110, labeled as PNC-2, lysed and subjected toimmunoprecipitation with anti-Ha-ras. These immunoprecipitates were thenblotted with anti-raf (lane 1), anti-MEK (lane 3), anti-JNK (lane 5) andanti-MAPK (lane 7). As can be seen in this figure, raf, MEK, JNK andMAPK all co-precipitate with Ha-Val 12-ras-p21. On the other hand, inthe presence of the two inhibitory peptides, none of these proteinsprecipitated with Val 12-ras-p21 although there is still some binding toraf.

The same experiment, the results of which are shown in FIG. 4A wasperformed on oocytes that were induced to mature with insulin. FIG. 4Bshows blots for raf (A), MEK (B), JNK (C) and MAPK (D). The first lanefor each of these four sets presents the results for the blots of wholecell lysate to demonstrate the presence of each protein. The second lanein each set of blots shows the results of blotting for each protein inthe anti-Ha-ras-p21 immunoprecipitate. As can be seen in this figure,only raf co-precipitates with endogenous Ha-ras-p21 in the oocytes. Thusoncogenic, but not activated wild-type, ras-p21 forms a large complexwith vital mitogenic signal transducing proteins and induces activationof raf-MEK-MAP kinase (MAPK or ERK) and JNK-jun pathways whileinsulin-activated wild-type p21 (at least the Ha-ras form) forms acomplex only with raf.

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What is claimed is:
 1. A replication incompetent Adenovirus (AdV) vectorcomprising a promoter sequence operably linked to a nucleotide sequenceencoding a peptide, wherein the peptide comprises at least about tencontiguous amino acids of the amino acid sequence: TIEDSYRKQVVID (SEQ IDNO: 2), or an analog or derivative thereof.
 2. A method of treating apatient suffering from cancer that expresses oncogenic ras-p21 protein,said method comprising administering to the patient, a therapeuticallyeffective amount of an AdV vector, said AdV vector comprising a promotersequence operably linked to a nucleotide sequence encoding an isolatedpeptide, wherein the isolated peptide comprises the amino acid sequenceTIEDSYRKQVVID (SEQ ID NO: 2)
 3. A method of inducing phenotypicreversion of cancerous cells to non-cancerous cells in a subject,wherein said cancerous cells express oncogenic ras-p21 protein, saidmethod comprising administering to the subject, a therapeuticallyeffective amount of an AdV vector, said AdV vector comprising a promotersequence operably linked to a nucleotide sequence encoding an isolatedpeptide, wherein the isolated peptide comprises the amino acid sequenceTIEDSYRKQVVID (SEQ ID NO: 2).
 4. The method of claim 3 wherein thecancerous cells that express oncogenic ras-p21 protein are colon cancercells, pancreatic cancer cells, non-small cell carcinoma of the lung,gastric cancer cells, bladder cancer cells or mesothelioma cells.